Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics / 解剖学报

ABSTRACT

Objective: To investigate the overexpression of paired box gene 6(Pax6) gene in mouse embryonic stem cells and obtain cell line, which is the basis for further differentiation of Pax6/mouse embryonic stem cells (mESCs). Methods: mESCs were cultured in vitro, and the recombinant vector pEFla-Pax6-IRES-AcGFP and the empty vector pEFlot-IRES-AcGFP were transfected into mESCs by liposome method respectively. The cells were screened by G418 gradient and fluorescent protein. The expression of Pax6 was detected by RT-PCR, immunofluorescence and Western blotting and the proportion of Pax6/mESCs positive cells was detected by flow cytometry. The obtained cell line was detected by cell immunofluorescence for stem cell markers stage specific embryonic antigen 1 (SSEA1) and octamer binding transcription factor 4(0CT4), and the pluripotency was detected by alkaline phosphatase staining. Pax6/mESCs cells were subcutaneously transplanted, and the grafts were observed by HE staining to observe their differentiation ability. Results: Pax6 was successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs was obtained, and the flow rate showed positive rate about 90%. Immunofluorescence showed that stem cell markers SSEA1 and 0CT4 were positively expressed and alkaline phosphatase stained cells were stained brownish black, and transplantation in vivo could differentiate into three germ layers. Conclusion: Pax6 is successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs is obtained and shows the good stem cell characteristics.